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Journal of Clinical Microbiology, August 2007, p. 2626-2634, Vol. 45, No. 8
0095-1137/07/$08.00+0     doi:10.1128/JCM.02501-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

High-Throughput, Sensitive, and Accurate Multiplex PCR-Microsphere Flow Cytometry System for Large-Scale Comprehensive Detection of Respiratory Viruses{triangledown} ,{dagger}

Wai-Ming Lee,1* Kris Grindle,1 Tressa Pappas,1 David J. Marshall,3 Michael J. Moser,3 Edward L. Beaty,3 Peter A. Shult,2 James R. Prudent,3 and James E. Gern1

Department of Pediatrics and Medicine,1 Wisconsin State Laboratory of Hygiene, University of Wisconsin, Madison, Wisconsin,2 EraGen Biosciences Incorporated, Madison, Wisconsin3

Received 13 December 2006/ Returned for modification 5 March 2007/ Accepted 20 April 2007

Human respiratory viruses are a diverse group of pathogens composed of hundreds of virus strains, and this presents a major challenge for diagnostic laboratories. To efficiently detect numerous viruses in a large epidemiologic study, we developed a fast, multitarget, sensitive, and specific assay named the Respiratory MultiCode-PLx Assay (RMA). The RMA utilizes improved multiplex PCR chemistry (EraGen MultiCode-PLx technology) coupled with high-throughput microsphere flow cytometry (Luminex). Eighteen sets of virus-specific multiplex PCR primers were developed based on the conserved sequences of all available respiratory-virus sequences for eight distinct groups: human rhinovirus (HRV), respiratory syncytial virus (RSV), parainfluenza virus (PIV), influenza virus (InfV), metapneumovirus, adenovirus (Ad), coronavirus, and enterovirus. Each primer set detected 20 cDNA copies of the intended target per sample and had no reaction with 60,000 copies of human genomic DNA. The accuracy and sensitivity of the RMA for detecting respiratory viruses in human samples were tested with two sets of clinical specimens. First, 101 nasal-wash specimens that were positive for HRV, RSV, InfV, PIV, or Ad by traditional techniques were reanalyzed by RMA, and all target viruses were detected with an overall sensitivity of 94% and specificity of 99%. Second, 103 nasal-wash samples from 5-year-old children with asthma and respiratory symptoms were analyzed; RMA detected viruses in 74 specimens (71.8%) compared to only 24 (23.3%) by traditional culture and immunofluorescent-staining techniques. These results show that RMA is an accurate, sensitive, and practical test for respiratory-virus infections.

* Corresponding author. Mailing address: University of Wisconsin Hospital, H4/469, 600 Highland Ave., Madison, WI 53792-9988. Phone: (608) 261-1387. Fax: (608) 265-9833. E-mail: wlee5{at}wisc.edu

{triangledown} Published ahead of print on 30 May 2007.

{dagger} Supplemental material for this article may be found at http://jcm.asm.org.

Journal of Clinical Microbiology, August 2007, p. 2626-2634, Vol. 45, No. 8
0095-1137/07/$08.00+0     doi:10.1128/JCM.02501-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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